G-protein-conjugated receptor having altered ligand affinity, and use thereof

ABSTRACT

A modified G-protein-coupled receptor (GPCR), having modified ligand affinity is provided by binding a G-protein-coupled receptor to a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1. Furthermore, agonists for or antagonists against the modified GPCR are screened using a transformant in which the modified GPCR has been expressed. This makes it possible to provide a technique for analyzing the function of many putative GPCRs whose entities have not been clarified.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a divisional application of U.S. Ser. No.12/451,558, filed Nov. 18, 2009, now U.S. Pat. No. 8,173,378, which isthe 35 U.S.C. §371 national stage of PCT application PCT/JP2008/059459,filed May 22, 2008, which claims benefit of Japanese Application2007-137275, filed May 23, 2007, the disclosures of all of which areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to G-protein-coupled receptors (GPCRs)having modified ligand affinity and use thereof and, more particularly,the present invention relates to a GPCR having ligand affinity modifiedby forming a complex with a particular protein and use thereof.

BACKGROUND ART

Many reactions in living organisms are caused by entrance ofextracellular information into cells and by propagation of theinformation in the cells. Membrane receptors serve as mediators throughwhich extracellular information is transmitted into cells. Among them,GPCRs having seven transmembrane domains is well known as a majorcategory of membrane receptors.

When a ligand (such as amino acids, peptides or amines) binds to a GPCR,the GPCR transmits its information into a cell via a trimeric G protein.G proteins coupled to GPCRs are classified into Gs, Gi, Gq, and thelike, which activate/inactivate different effector pathways (e.g., cAMPpathway, cGMP pathway, and phospholipase C pathway), respectively. Forexample, α₁-adrenergic receptor is mainly coupled to Gq protein topromote phospholipase C system, which produces diacylglycerol andinositol trisphosphate, thus increasing intracellular Ca²⁺.α₂-adrenergic receptor is mainly coupled to Gi protein to suppressadenylate cyclase system, thus decreasing cAMP. Further, β-adrenergicreceptor is mainly coupled to Gs protein to promote adenylate cyclasesystem, which thus increases cAMP.

GPCRs widely occur and function in our body. For example, α₁-adrenergicreceptors exist peripherally in blood vessel, prostate, and produce thecontractions. Further, α₁-adrenergic receptors are known to function incentral nervous system. β-adrenergic receptors in heart and fat tissueplay important roles in heart rate and lipolysis.

GPCRs and their signal transduction systems are known not only tocontrol the physiological homeostasis in our body but also to beinvolved in pathophysiological status of various diseases. Therefore, inorder to treat the diseases it will be very significant to identify theGPCRs which are related to the diseases and then to develop theirspecific drugs (such as agonists or antagonists).

-   Non Patent Literature 1-   Muramatsu, I. et al. Br. J. Pharmacol., 99: 197 (1990)-   Non Patent Literature 2-   Muramatsu, I. et al. Pharmacol. Commun., 6: 23 (1995)-   Non Patent Literature 3-   Morishima, S. et al. J. Urol. 177: 377-381 (2007)-   Non Patent Literature 4-   Molenaar, P. and Parsonage, W. A. Trends Pharmacol. Sci., 26:    368-375 (2005)-   Non Patent Literature 5-   Samaha, A. N. et al., J. Neurosci. 27: 2979-2986 (2007)

SUMMARY OF INVENTION

There has been significant progress in GPCR research, whereby a largenumber of GPCRs have been identified. However, there still exist severalputative GPCRs whose phenotypes are identified but whose entities areyet unknown. For example, α₁-adrenergic receptors are now classifiedinto three subtypes (α_(1A), α_(1B) and α_(1D)) based on their distinctgenes. However, in addition to the classical α₁-adrenergic receptors,the presence of an additional subtype (α_(1L)) which shows differentpharmacological profile (phenotype) from the classical subtypes has beenproposed. The α_(1L)-subtype has significantly lower affinity for arepresentative α₁ blocker (prazosin) than α_(1A)-, α_(1B)- andα_(1D)-subtypes. The α_(1L)-subtype can be detected only in intactstrips or segments of native tissues but not be identified in theirtissue homogenates (see Non Patent Literatures 1 to 3). Further,subtypes of β₁ adrenaline receptor (β_(1H) and β_(1L)) that differ inphenotype are known to be expressed from the same genes (see Non PatentLiterature 4). Such a subtype only has its phenotype known, and has itsentity unknown. The similar cases may be also pointed out for dopaminereceptors, muscarinic receptors, or endothelin receptors (see Non PatentLiterature 5 and the like). However, the underlying mechanisms fordifferent phenotype formation of the same gene product have not yetknown.

The present invention has been made in view of this problem, and it isan object of the present invention to provide a technique for analyzingthe function of a G-protein-coupled receptor whose entity has not beenclarified.

A protein complex according to the present invention is characterized bybinding of a GPCR to (1) a polypeptide consisting of an amino-acidsequence of SEQ ID NO: 1; (2) a polypeptide (i) consisting of anamino-acid sequence of SEQ ID NO: 1 with a deletion, insertion,substitution, or addition of one or several amino acids, and (ii) havingactivity to modify ligand affinity of a GPCR with which the polypeptidehas formed a complex; (3) a polypeptide encoded by a polynucleotideconsisting of a nucleotide sequence of SEQ ID NO: 2; (4) a polypeptide(i) encoded by a polynucleotide consisting of a nucleotide sequence ofSEQ ID NO: 2 with a deletion, insertion, substitution, or addition ofone or several nucleotides, and (ii) having activity to modify ligandaffinity of a GPCR with which the polypeptide has formed a complex; (5)a polypeptide (i) encoded by a polynucleotide capable of hybridizingunder stringent conditions with a polynucleotide consisting of asequence complementary to a nucleotide sequence of SEQ ID NO: 2 and (ii)having activity to modify ligand affinity of a GPCR with which thepolypeptide has formed a complex; or (6) a polypeptide (i) coded for bya polynucleotide having a sequence identity of 70% or higher with apolynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and(ii) having activity to modify ligand affinity of a GPCR with which thepolypeptide has formed a complex.

A method according to the present invention for producing a proteincomplex is characterized by including the step of causing a GPCR and apolypeptide to coexist on a lipid membrane, the polypeptide being (1) apolypeptide consisting of an amino-acid sequence of SEQ ID NO: 1; (2) apolypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1with a deletion, insertion, substitution, or addition of one or severalamino acids, and (ii) having activity to modify ligand affinity of aGPCR with which the polypeptide has formed a complex; (3) a polypeptideencoded by a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2; (4) a polypeptide (i) encoded by a polynucleotide consistingof a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,substitution, or addition of one or several nucleotides, and (ii) havingactivity to modify ligand affinity of a GPCR with which the polypeptidehas formed a complex; (5) a polypeptide (i) encoded by a polynucleotidecapable of hybridizing under stringent conditions with a polynucleotideconsisting of a sequence complementary to a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex; or (6) a polypeptide(i) coded for by a polynucleotide having a sequence identity of 70% orhigher with a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex.

With this feature, the present invention can modify the ligand affinityof a GPCR. That is, the method according to the present invention forproducing a protein complex can also be a method for modifying theaffinity of a GPCR for its ligands.

A lipid membrane according to the present invention is characterized bycontaining the protein complex. A method according to the presentinvention for producing the lipid membrane is characterized by includingthe step of causing a GPCR and a polypeptide to coexist on the lipidmembrane, the polypeptide being (1) a polypeptide consisting of anamino-acid sequence of SEQ ID NO: 1; (2) a polypeptide (i) consisting ofan amino-acid sequence of SEQ ID NO: 1 with a deletion, insertion,substitution, or addition of one or several amino acids, and (ii) havingactivity to modify ligand affinity of a G-protein-coupled receptor withwhich the polypeptide has formed a complex; (3) a polypeptide encoded bya polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2;(4) a polypeptide (i) encoded by a polynucleotide consisting of anucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,substitution, or addition of one or several nucleotides, and (ii) havingactivity to modify ligand affinity of a GPCR with which the polypeptidehas formed a complex; (5) a polypeptide (i) encoded by a polynucleotidecapable of hybridizing under stringent conditions with a polynucleotideconsisting of a sequence complementary to a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex; or (6) a polypeptide(i) coded for by a polynucleotide having a sequence identity of 70% orhigher with a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex.

Furthermore, a transformant according to the present invention ischaracterized by containing the protein complex. A method according tothe present invention for producing the transformant is characterized byincluding the step of expressing the protein complex, and preferablyincludes the step of introducing, into a cell, a gene encoding a GPCRand a gene encoding the polypeptide.

With this feature, the present invention makes it easy to analyze thefunction of a GPCR having modified ligand affinity.

A method according to the present invention for screening agonists orantagonists of a GPCR having modified ligand affinity is characterizedby including the steps of: [I] generating a protein complex by causing aGPCR and a polypeptide to coexist on a lipid membrane; and [II]incubating the protein complex together with a candidate factor, thepolypeptide being (1) a polypeptide consisting of an amino-acid sequenceof SEQ ID NO: 1; (2) a polypeptide (i) consisting of an amino-acidsequence of SEQ ID NO: 1 with a deletion, insertion, substitution, oraddition of one or several amino acids, and (ii) having activity tomodify ligand affinity of a GPCR with which the polypeptide has formed acomplex; (3) a polypeptide encoded by a polynucleotide consisting of anucleotide sequence of SEQ ID NO: 2; (4) a polypeptide (i) encoded by apolynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 witha deletion, insertion, substitution, or addition of one or severalnucleotides, and (ii) having activity to modify ligand affinity of aGPCR with which the polypeptide has formed a complex; (5) a polypeptide(i) encoded by a polynucleotide capable of hybridizing under stringentconditions with a polynucleotide consisting of a sequence complementaryto a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity tomodify ligand affinity of a GPCR with which the polypeptide has formed acomplex; or (6) a polypeptide (i) coded for by a polynucleotide having asequence identity of 70% or higher with a polynucleotide consisting of anucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modifyligand affinity of a GPCR with which the polypeptide has formed acomplex.

A method according to the present invention for producing a transformantexpressing a GPCR having modified ligand affinity is characterized byincluding the step of inhibiting expression of a polypeptide in the cellin which a GPCR has been expressed, the polypeptide being (1) apolypeptide consisting of an amino-acid sequence of SEQ ID NO: 1; (2) apolypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1with a deletion, insertion, substitution, or addition of one or severalamino acids, and (ii) having activity to modify ligand affinity of aGPCR with which the polypeptide has formed a complex; (3) a polypeptideencoded by a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2; (4) a polypeptide (i) encoded by a polynucleotide consistingof a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,substitution, or addition of one or several nucleotides, and (ii) havingactivity to modify ligand affinity of a GPCR with which the polypeptidehas formed a complex; (5) a polypeptide (i) encoded by a polynucleotidecapable of hybridizing under stringent conditions with a polynucleotideconsisting of a sequence complementary to a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex; or (6) a polypeptide(i) coded for by a polynucleotide having a sequence identity of 70% orhigher with a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex.

With this feature, the present invention makes it easy to analyze thefunction of a GPCR having modified ligand affinity, and can modify theligand affinity of a GPCR. That is, the present invention can also be amethod for modifying the affinity of a GPCR for its ligands.

The producing method according to the present invention is preferablysuch that the polypeptide is an endogenous protein, and that the step ofinhibiting the expression of the polypeptide is performed according toan RNAi method. Further, the cell may be a transformant expressing anexogenous GPCR.

A method according to the present invention for screening agonists orantagonists of a GPCR having modified ligand affinity is characterizedby including the steps of: [I] inhibiting expression of a polypeptide inthe cell in which a GPCR has been expressed; and [II] incubating thecell together with a candidate factor, the polypeptide being (1) apolypeptide consisting of an amino-acid sequence of SEQ ID NO: 1; (2) apolypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1with a deletion, insertion, substitution, or addition of one or severalamino acids, and (ii) having activity to modify ligand affinity of aGPCR with which the polypeptide has formed a complex; (3) a polypeptideencoded by a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2; (4) a polypeptide (i) encoded by a polynucleotide consistingof a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,substitution, or addition of one or several nucleotides, and (ii) havingactivity to modify ligand affinity of a GPCR with which the polypeptidehas formed a complex; (5) a polypeptide (i) encoded by a polynucleotidecapable of hybridizing under stringent conditions with a polynucleotideconsisting of a sequence complementary to a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex; or (6) a polypeptide(i) coded for by a polynucleotide having a sequence identity of 70% orhigher with a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex. It is preferable thatthe screening method according to the present invention further includethe step of measuring an intracellular Ca²⁺ concentration or the step ofmeasuring metabolism of intracellular inositol phosphate.

In the present invention, it is preferable that the GPCR constitutingthe protein complex be an adrenergic receptor, a dopamine receptor, amuscarinic receptor, or an endothelin receptors, and the adrenergicreceptors may be α-receptor or β-receptor. Further, it is preferablethat the dopamine receptor be a D2 receptor. It is preferable that theα-receptor be an α₁-receptor, and it is more preferable that theα₁-receptor be an α_(1A)-subtype. In a preferred embodiment, a proteincomplex according to the present invention is α_(1L)-subtype orβ_(1L)-subtype of adrenergic receptors.

For a fuller understanding of the nature and advantages of theinvention, reference should be made to the ensuing detailed descriptiontaken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1

FIG. 1 shows saturation binding curves for [³H]-silodosin in α_(1L)cells (whole cell binding experiment).

FIGS. 2( a) and (b)

(a) of FIG. 2 shows competition-binding curves for prazosin in α_(1L)cells and α_(1A) cells (whole cell binding experiment). (b) of FIG. 2shows competition-binding curves for prazosin in homogenates of α_(1L)cells and α_(1A) cells. Dashed line with closed squares: Acompetition-binding curve for prazosin in whole α_(1L) cells wasdescribed for comparison against the curves in homogenates.

DESCRIPTION OF EMBODIMENTS

[1: Protein Complex]

The present invention provides a protein complex of a GPCR and aparticular polypeptide. When used in the present specification, the term“complex” means an integrated combination of a plurality of substances,and “complex formation” and “integration” are used interchangeably. Itshould be noted that a plurality of substances forming a complex onlyneed to interact with each other in close proximity and may or may notbind to each other. In a preferred embodiment, a protein complexaccording to the present invention is such a state that a GPCR and aparticular polypeptide interact with each other in close proximity and,as a result, functions as a GPCR having modified ligand affinity.

The present specification describes the present invention by taking anadrenergic receptor (in particular, of α_(1A)-subtype) as an example ofa GPCR whose ligand affinity is modified by interacting with aparticular polypeptide. However, a person skilled in the art who hasread the present specification would easily understand that a GPCRconstituting the present invention is not limited to an adrenergicreceptor. Further, a person skilled in the art could easily obtaininformation on the sequence of a GPCR constituting the presentinvention.

Adrenergic receptors are well known to mediate the functions of theautonomic nervous system (sympathetic nervous system). Binding ofadrenaline, noradrenaline, or the like to the adrenergic receptor causesvarious physiological responses such as contractions of vascular smoothmuscle, increases in blood pressure and heart rate, dilation of thepupil, and an increase in blood glucose level.

The progress in research and the development of a new variety of drugshave made clear that adrenergic receptors are classified into varioustypes. In the past, adrenergic receptors were classified into α-receptorand β-receptor according to their difference in reactivity to drugs suchas isoproterenol and phentolamine, and further classified intopharmacological subtypes (α₁-receptor, α₂-receptor, and β-receptor).These receptors are known to exhibit different distributions in eachtissue and to play different functions. For example, the α₁-adrenergicreceptors are known to play as an important mediator causingcontractions of vascular smooth muscle, prostate, and the like, and alsoknown to be involved in the regulation of consciousness and emotion incentral nervous system.

Now that the mapping of the human genome has been finished, thestructures and functions of many proteins can be shown on a geneticlevel. In α₁-adrenergic receptors, three subtypes (α_(1A), α_(1B), andα_(1D)) were identified according to their distinct genes. Theseclassical subtypes are known to coincide well with pharmacologicallyidentified subtypes. In this way, it may be considered that one receptorsubtype is basically originated from one distinct gene.

However, it has been long pointed out that a unique α₁-adrenergicreceptor occurs and functions in some tissues of our body. Because ofits low affinity for a representative α₁ blocker (prazosin), the uniquesubtype has been called “α_(1L)-subtype”, although the correspondinggene has not been yet cloned.

Table 1 shows the classification and drug selectivity of α₁-adrenergicreceptors.

TABLE 1 Classification and Drug Selectivity of α₁-adrenergic ReceptorsAffinity (pKb) Subtypes silodosin^(1, 3, 4, 5) tamsulosin^(1, 3, 4)prazosin^(1, 2, 3, 4, 5) RS- 17053^(2, 4) BMY 7378^(4, 5) α1A 10.7-9.510.4-9.9 10.6-9.3  9.1-8.4 6.9-5.6 α1L 10.7-9.5 10.4-9.9 8.3-7.6 6.36.9-5.6 α1B 8.1 9.3 10.6-10.1 7.8 7.4 α1D 8.6 9.9 10.1-9.9  7.8 9.1

The effects of the compounds shown in Table 1 are based on the followingliteratures:

-   1. Muramatsu, I. et al. Pharmacol. Commun., 6: 23 (1995)-   2. Ford, A P. et al. Mol. Pharmacol., 49: 209 (1996)-   3. Murata, S. et al. J. Urol., 164: 578 (2000)-   4. Hiraizumi-Hiraoka, Y. et al. J. Pharmacol. Exp. Ther., 310: 995    (2004)-   5. Murata, S. et al. Br. J. Pharmacol., 127: 19 (1999)

In bioassay studies, the α_(1L)-subtype has been clearly demonstrated asa functional receptor in the lower urinary tract systems of human andother mammals. The α_(1L)-subtype was also identified, if the intactsegments of native tissues (e.g. human prostate). However, theα_(1L)-subtype was not detected by a conventional binding experimentconducted with homogenized tissue. The fact that α_(1L)-subtype is notdetected in homogenized tissue means that purification of theα_(1L)-subtype from tissue is very difficult. Unless the entity of theα_(1L)-subtype is clarified, it will be very difficult to analyze thefunction of α_(1L)-adrenergic receptor, in particular, to developα_(1L)-selective drugs (such as agonists or antagonists).

The inventors assumed that the α_(1L)-subtype, whose entity is unknown,is constituted by binding of some sort of ancillary molecule to analready known subtype (probably, α_(1A)-subtype). That is, the inventorsassumed that the interaction between the subtype molecule and themolecule ancillary thereto, which constitute the α_(1L)-subtypetogether, is dissolved by homogenizing tissue and, as a result, theα_(1L)-subtype changes its properties into those of the α_(1A)-subtype.In the result, the inventors found that the pharmacological profile ofα_(1L)-subtype was converted to that of α_(1A)-subtype upon tissuehomogenization. Furthermore, as a result of their diligent studies, theinventors confirmed that a particular protein binds to theα_(1A)-subtype, and that coexpression of the protein with theα_(1A)-subtype in a cultured cell leads to expression of a phenotype ofα_(1L)-subtype. That is, the inventors found that the α_(1L)-subtypeconsists of a protein complex, identified a protein constituting thecomplex, and thereby accomplished the present invention.

In one embodiment, the present invention provides a protein complex thatforms an α_(1L)-subtype of adrenergic receptor. That is, a proteincomplex according to the present embodiment is an α_(1L)-subtype ofadrenergic receptor. The present embodiment makes it possible to providetreatment for any disease associated with an α_(1L)-subtype whose entityhas been clarified.

A polypeptide constituting the protein complex according to the presentembodiment is already publicly known as a CRELD (cysteine-rich withEGF-like domains) 1α protein, whose missense mutation is pointed out asbeing associated with an atrioventricular septal defect (Gene 293: 47-57(2002), Am. J. Hum. Genet. 72: 1047-1052 (2003)). Information on thesequence of the polypeptide is provided as NCBI Accession No.NM_(—)015513 and, in the present specification, represented as SEQ IDNO: 1 (amino-acid sequence) and SEQ ID NO: 2 (nucleotide sequence). Thatis, the polypeptide constituting the protein complex according to thepresent embodiment may be a polypeptide consisting of an amino-acidsequence of SEQ ID NO: 1, or may be a polypeptide coded for by apolynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2.

The polypeptide constituting the protein complex according to thepresent embodiment is not limited to a polypeptide consisting of anamino-acid sequence of SEQ ID NO: 1, and may be a mutant polypeptideretaining the activity of the original polypeptide. An example of such amutant polypeptide is a polypeptide (i) consisting of an amino-acidsequence of SEQ ID NO: 1 with a deletion, insertion, substitution, oraddition of one or several amino acids, and (ii) having the activity toform a complex with a GPCR and modify the ligand affinity of the GPCR.

With technical common sense in the field, a person skilled in the artcould easily produce a mutant polypeptide, in the amino-acid sequence ofa particular polypeptide with a deletion, insertion, substitution, oraddition of one or several amino acids. Further, based on thedescriptions in the present specification and the technical commonsense, a person skilled in the art could easily confirm whether or notthe mutant polypeptide retains the same activity as the originalpolypeptide.

Further, the polynucleotide encoding the polypeptide constituting theprotein complex according to the present embodiment is not limited to apolynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2, andonly needs to be a mutant polynucleotide encoding a polypeptideretaining the activity to modify the ligand affinity of a GPCR withwhich the polypeptide has formed a complex. An example of such a mutantpolynucleotide is, but is not limited to, (1) a polynucleotideconsisting of a nucleotide sequence of SEQ ID NO: 2 with a deletion,insertion, substitution, or addition of one or several nucleotides, (2)a polynucleotide capable of hybridizing under stringent conditions witha polynucleotide consisting of a sequence complementary to a nucleotidesequence of SEQ ID NO: 2, or (3) a polynucleotide having a sequenceidentity of 70% or higher, preferably 80% or higher, or more preferably85% or higher, with a polynucleotide consisting of a nucleotide sequenceof SEQ ID NO: 2.

With technical common sense in the field, a person skilled in the artcould easily produce: a mutant polypeptide, in the amino-acid sequenceof a particular polynucleotide with a deletion, insertion, substitution,or addition of one or several amino acids; a mutant polynucleotidecapable of hybridizing under stringent conditions with a particularpolynucleotide; or a mutant polynucleotide having a sequence identity of70% or higher with a particular polynucleotide. Further, based on thedescriptions in the present specification and the technical commonsense, a person skilled in the art could easily confirm whether or not apolypeptide coded for by the mutant polynucleotide retains the sameactivity as a polypeptide coded for by the original polynucleotide.

When used in the present specification, the term “stringenthybridization conditions” means overnight incubation at 42° C. in ahybridization solution (containing 50% formamide, 5×SSC [150 mM of NaCl,15 mM of trisodium citrate], 50 mM of sodium phosphate (with a pH of7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml ofsheared and denatured salmon sperm), followed by washing of a filter atapproximately 65° C. in 0.1×SSC.

A specific procedure for hybridization only needs to be performedaccording to a method well known in the field (e.g., a method describedin “Molecular Cloning: A Laboratory Manual, 3rd ed., J. Sambrook and D.W. Russell ed., Cold Spring Harbor Laboratory, NY (2001)” [which isincorporated by a reference to the present specification]).

The present invention thus far has been described by taking anadrenergic receptor as an example of a GPCR constituting a proteincomplex according to the present invention. However, a protein complexaccording to the present invention only needs to be a complex of a GPCRand (1) a polypeptide consisting of an amino-acid sequence of SEQ ID NO:1; (2) a polypeptide (i) consisting of an amino-acid sequence of SEQ IDNO: 1 with a deletion, insertion, substitution, or addition of one orseveral amino acids, and (ii) having activity to modify ligand affinityof a GPCR with which the polypeptide has formed a complex; (3) apolypeptide encoded by a polynucleotide consisting of a nucleotidesequence of SEQ ID NO: 2; (4) a polypeptide (i) encoded by apolynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 witha deletion, insertion, substitution, or addition of one or severalnucleotides, and (ii) having activity to modify ligand affinity of aGPCR with which the polypeptide has formed a complex; (5) a polypeptide(i) encoded by a polynucleotide capable of hybridizing under stringentconditions with a polynucleotide consisting of a sequence complementaryto a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity tomodify ligand affinity of a GPCR with which the polypeptide has formed acomplex; or (6) a polypeptide (i) coded for by a polynucleotide having asequence identity of 70% or higher with a polynucleotide consisting of anucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modifyligand affinity of a GPCR with which the polypeptide has formed acomplex. That is, the GPCR constituting the protein complex is notlimited to an adrenergic receptor, but may be dopamine receptors,muscarinic receptors, or endothelin receptors. Further, a person skilledin the art could easily obtain, from a publicly-known database,information on the sequence of a GPCR constituting the presentinvention, and therefore could easily produce a desired protein complex,based on the descriptions in the present specification and the technicalcommon sense.

[2. Lipid Membrane Containing a Protein Complex]

The present invention also provides a method for producing a proteincomplex of a GPCR and a particular polypeptide. The method according tothe present invention for producing a protein complex is characterizedby including the step of causing a GPCR and a particular polypeptide tocoexist on a lipid membrane. That is, the present invention provides alipid membrane containing a protein complex and a method for producingthe same.

A lipid membrane according to the present invention is characterized bycontaining such a protein complex as described above. The lipid membraneaccording to the present invention may be a naturally-occurring lipidmembrane, or may be an artificial lipid membrane. In cases where thelipid membrane is a naturally-occurring lipid membrane, the lipidmembrane is intended to be a biological membrane. In cases where thelipid membrane is an artificial lipid membrane, the lipid membrane isintended to be a lipid planar membrane or liposome.

In one aspect of the present invention, a lipid membrane can be abiological membrane of a transformant having introduced thereinto apolynucletoide encoding a polypeptide constituting a protein complexaccording to the present invention. Such a transformant can be obtainedby introducing an expression vector containing the polynucleotide into aliving organism so that the polypeptide is expressed in the form of abiological membrane. It should be noted that the living organism for useas the transformant may be a prokaryotic organism or a eucaryoticorganism.

In another aspect of the present invention, a lipid membrane can be alipid bilayer containing a polypeptide constituting a protein complex ofthe present invention. The lipid bilayer is a membranous structurecomposed of two layers of polar lipid (in particular, phospholipid). Thelipid bilayer structure is stabilized as a two-dimensional structurewhen it takes the form of a sphere, but can be a planar structure if itsend is isolated from a water molecule. When used in the presentspecification, the term “liposome” means a spherical lipid bilayer thatis made artificially, and the term “lipid planar membrane” means aplanar lipid bilayer that is made artificially. In the field, anartificial lipid bilayer is used in in vitro measurement of the activityof a membrane protein (e.g., a channel protein). In this way, a personskilled in the art could easily produce a lipid planar membrane andcause the lipid planar membrane to retain a protein (polypeptide) ofinterest. Further, liposome is a lipid artificial membrane that isreferred to also as “vesicle”, and can be produced by beating up asuspension of lipid (e.g., phospholipid) and then subjecting it toultrasonication. In the field, researches have been widely conductedwith liposome as a cell membrane model or as a means of drug deliverysystem (DDS). In this way, a person skilled in the art could easilyproduce liposome and cause the liposome to retain a protein(polypeptide) of interest.

In one embodiment, the present invention provides a lipid membranecontaining an α_(1L)-subtype of adrenergic receptor. As described above,the α_(1L)-subtype of adrenergic receptor is a protein complex of anα_(1A)-subtype molecule and a CRELD1α protein. The α_(1A)-subtype ofadrenergic receptor and the CRELD1α are both membrane-bound proteins,and, as described above, their interaction with each other is dissolvedby homogenization (membrane disruption). That is, their interaction witheach other is expressed by placing and retaining both of them on a lipidmembrane.

In cases where the lipid membrane according to the present embodiment isa biological membrane, a method according to the present embodiment forproducing a lipid membrane can also be a method for producing atransformant that expresses an α_(1L)-subtype of adrenergic receptor,and only needs to include the step of coexpressing an α_(1A)-adrenergicreceptor protein and a CRELD1α protein. Information on the sequence ofthe α_(1A)-subtype of adrenergic receptor is provided as NCBI AccessionNo. U03866 or NM_(—)000680 and, in the present specification,represented as SEQ ID NO: 3 (amino-acid sequence) and SEQ ID NO: 4(nucleotide sequence). That is, a method according to the presentembodiment for producing a protein complex only needs to include thestep of transforming a host with a vector containing a polynucleotideconsisting of a nucleotide sequence of SEQ ID NO: 2 and a vectorcontaining a polynucleotide consisting of a nucleotide sequence of SEQID NO: 4.

It is preferable that the vectors be each an expression vector having apolynucleotide of interest operably linked therewith. When used in thepresent specification, the term “operably linked” means that apolynucleotide encoding a peptide (or protein) of interest is undercontrol of a control region such as a promoter and is in such a formthat the peptide (or protein) can be expressed in a host. A procedurefor establishing a desired vector by “operably linking” a polynucleotideencoding a peptide of interest with an expression vector is well knownin the field. Further, a method for introducing an expression vectorinto a host is well known, too, in the field. Therefore, a personskilled in the art could easily generate a desired peptide in a host.

When used in the present specification, the term “transformant” meansnot only a cell, tissue, or an organ, but also an individual livingorganism. Examples of a living organism serving as an object oftransformation include, but are not limited to, various microorganisms,plants, and animals. It should be noted that a transformant according tothe present invention only needs to have introduced thereinto at least apolynucleotide encoding a polypeptide constituting a protein complexaccording to the present invention and express such polypeptides. Thatis, it should be noted that a transformant generated by means other thanan expression vector is encompassed, too, in the technical scope of thepresent invention. Further, although it is preferable that atransformant according to the present invention be stably expressing apolypeptide constituting a protein complex according to the presentinvention, the polypeptide of interest may be transiently expressed. Itshould be noted that use of a transformant coexistent with a proteincomplex according to the present embodiment makes it possible to screenan agonist for or antagonist against the complex by observing thebehavior of a second messenger in a host cell.

In cases where the lipid membrane according to the present embodiment isan artificial membrane, the method according to the present embodimentfor producing a lipid membrane can also be a method for producing alipid planer membrane or liposome containing an α_(1L)-subtype ofadrenergic receptor and, more specifically, only needs to include thestep of reconstituting an α_(1A)-adreneregic receptor protein and aCRELD1α protein on an artificial planer membrane. With common sense inthe field, a person skilled in the art could easily reconstitute amembrane protein on an artificial lipid membrane. Use of an artificiallipid membrane coexistent with a protein complex according to thepresent embodiment makes it possible to measure the ligand affinity ofthe complex.

[3. Use of a Lipid Membrane]

A person skilled in the art would easily understand that theabove-described method for producing a lipid membrane can be both amethod for producing a protein complex and a method for modifying theligand affinity of a GPCR. That is, the present invention provides amethod for modifying the affinity of a GPCR for its ligands.

Further, use of the lipid membrane according to the present inventionmakes it possible to screen an agonist for or antagonist against a GPCRhaving modified ligand affinity. That is, the present invention providesa method for screening agonists for or antagonists against a GPCR havingmodified ligand affinity.

The screening method according to the present invention is characterizedby including the steps of generating such a protein complex as describedabove on a lipid membrane; and incubating the protein complex togetherwith a candidate factor. When used in the present specification,“incubating” means causing a plurality of substances to coexist andputting them in such a state that they make sufficient contact with eachother.

In a preferred embodiment, the screening method according to the presentinvention includes the step of incubating, together with a candidatefactor, a transformant containing a GPCR of interest (e.g., a cellhaving α_(1L)-subtype of adrenergic receptor expressed therein). Thescreening method according to the present embodiment makes it possibleto screen agonists for or antagonists against a GPCR of interest (e.g.,an α_(1L)-subtype of adrenergic receptor) by measuring the fluctuationof a second messenger in a cell (e.g., the amount of cAMP in cases wherethe G protein is Gs protein or Gi protein or the concentrations ofinosine triphosphate and Ca²⁺ in cases where the G protein is Gqprotein).

The agonists for α_(1L)-subtype of adrenergic receptor can be used as adrug for urinary incontinence, a mydriatic drug, a drug for glaucoma,and a drug for central stimulation. The antagonists againstα_(1L)-subtype of adrenergic receptor can be used as a drug for urinarydisturbance, a drug for Raynaud's disease, a drug for microcirculatoryfailure, an antihypertensive drug, a central depressant, a drug forimprovement of renal blood flow, and a diuretic drug. Further, sinceβ_(1L)-subtype of adrenergic receptor is known to be associated withheart disease and dopamine D2 receptor and muscarinic receptor are knownto be associated with central nervous system disease, agonists orantagonists thereto are useful, too.

[4. Knockdown Cell and Use Thereof]

The present invention also provides a transformant in which apolypeptide that forms a protein complex with a GPCR has been knockeddown and a method for producing the same. A method according to thepresent invention for producing a transformant is characterized byincluding the step of inhibiting expression of an endogenouspolypeptide.

For example, the CRELD1α protein, which is a polypeptide that forms acomplex with a GPCR, is expressed in various cells. Therefore, atransformant in which expression of an endogenous CRELD1α protein hasbeen inhibited is very useful as a control cell that is used inanalyzing the function of the protein complex or screening a targetcompound (e.g., an agonist or antagonist). The knockdown cell, describedin this section, may be used alone, but is preferably used incombination with the above-described protein complex or theabove-described lipid membrane containing the protein complex. It shouldbe noted that the GPCR expressed in the object cell may be endogenous orexogenous.

In one aspect, the present invention can include the step of inhibitingexpression of an endogenous CRELD protein in a cell in which a GPCR hasbeen expressed. This makes it possible to modify the ligand affinity ofthe GPCR expressed in the cell. That is, the method according to thepresent invention for producing a transformant can be both a method forproducing a transformant expressing a GPCR having modified ligandaffinity and a method for modifying the affinity of a GPCR for itsligand. In one embodiment, the present invention can include the step ofinhibiting expression of an endogenous CRELD1α protein in a cellexpressing an α_(1L)-subtype of adrenergic receptor. This makes itpossible to modify a cell indicative of a phenotype of α_(1L)-subtype tobe a cell indicative of a phenotype of α_(1A)-subtype.

It is preferable that an RNAi method be used as a technique forinhibiting expression of an endogenous CRELD1α protein. The RNAi methodis a technique well known in the field; for example, expression of anendogenous CRELD1α protein can be successfully inhibited by introducing,into a cell of interest, an oligonucleotide consisting of a nucleotidesequence of SEQ ID NO: 5. In the case of use of a vector forintroducing, into a cell of interest, an oligonucleotide consisting of anucleotide sequence of SEQ ID NO: 5, it is preferable that anoligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 6 beused as an antisense, without implying any limitation.

The present invention further provides a method, characterized by usingthe above-described transformant, for screening an agonist for orantagonist against a GPCR having modified ligand affinity.

The present invention is not limited to the description of theembodiments above, but may be altered by a skilled person within thescope of the claims. An embodiment based on a proper combination oftechnical means disclosed in different embodiments is encompassed in thetechnical scope of the present invention.

All the academic and patent literatures cited herein are incorporated byreferences to the present specification.

EXAMPLE 1. Structures

A human α_(1A)-adrenergic receptor gene (ADRA1A) was cloned from a humanprostate library with a PCR technique. The cloned gene had a wholelength of 1465 bp, and had an ORF sequence perfectly matching that ofthe human alpha-1A gene (NCBI Accession No. U03866 or NM_(—)000680),which has conventionally been reported. Further, sequences preceding andfollowing the ORF sequence matched those reported in Hirasawa et al.(1993).

A CRELD1α gene was cloned from a human prostate cDNA library. As aresult of sequence determination, the cloned gene matched that reportedin Rupp et al. (2002) (NCBI Accession No. NM_(—)015513).

Next, the coding region of the ADRA1A gene was subcloned into the EcoRIrestriction enzyme site of the multicloning site A of the pIRES(Clonetech, Catalog No. PT3266-5). Further, the coding region of theCRELD1α was subcloned into the XbaI restriction enzyme site of themulticloning site B of the pIRES. For the purpose of subcloning, each ofthe genes had a restriction enzyme adapter added to each end.

2. Production of a Transformant

The vector was amplified/purified in a conventional method, and thentransfected into CHO-K1 cells, which are cells of Chinese Hamsterovarian origin, with Lipofectamine 2000 (Invitrogen Corporation)according to the manufacturer's protocol. Two days after thetransfection, 1200 μg/ml of the antibiotic Geneticin (G418) were addedto the DMEM culture medium. Cells into which the vector had not beentransfected, i.e., cells subjected to the same operation withLipofectamine 2000 and the like expect that the vector had not beenadded, were completely annihilated at a G418 concentration of 1000μg/ml. The G418-resistant cells were diluted, suspended, and injecteddividedly into a 96-well plate so that an average of 0.5 cells was putin per well, and a single colony was chosen. This operation was repeatedthree times, whereby a stable clone (α_(1L) cells) having coexpressedthe ADRA1A gene and the CREDL1α gene was produced. Further, as controlgroups, the cells (α_(1A) cells) having stably expressed only the ADRA1Agene and the cells (CREDL1α cells) having stably expressed only theCREDL1α gene were produced.

3. Binding Experiment

The drugs used in the binding experiment and their proper chemical namesare as follows: silodosin, prazosin, tamsulosin, RS-17053(N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethanaminehydrochloride), BMY 7378(8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]8-azaspiro[4,5]decane7,9-dionedihydrochloride).

The stable clone was studied by a pharmacological binding method with[³H]-silodosin, which is a radioligand. An experiment was conductedusing a whole-cell binding method. That is, cells were scattered two tothree days before the experiment so that they are semi-confluent, washedtwice with ice-cold PBS, and then scraped by a scraper. The cells thuscollected were suspended again in a Krebs-HEPES solution, and thenincubated at 4° C. for four hours together with [³H]-silodosin andanother drug as needed. Thereafter, the cells were filtered/washed by aWhatmann GC/F filter pretreated with polyethylene imine, and theradioactivity was measured by a liquid scintillation counter. Thenon-specific binding was evaluated as binding in the presence of 30 μMphenotolamine.

[A] Saturation Binding Experiment According to the Whole-Cell Method

The binding of [³H]-silodosin of various concentrations (30 to 1000 pM)to the α_(1L) cells was examined (FIG. 1). FIG. 1 shows a saturationbinding curve obtained by the whole-cell method. The specific binding of[³H]-silodosin to the α_(1L) cells exhibited a maximum binding amount(Bmax) of 134 fmol/mg protein and a Kd value of 843 pM (pK_(D)=9.1).

[B] Competitive Binding Experiment

Silodosin is known to bind selectively to the α_(1A)- andα_(1L)-subtypes with a high affinity. The term “high affinity” heremeans a Kd value of 1 nM or less. In order to identify the pharmacologicproperties of receptors expressed in the α_(1L) cells, a competitivebinding experiments with prazosin at 300 pM [³H]-silodosin binding siteswere conducted using the whole-cell binding experiment method. Theα_(1A) cells were used as control cell line. [³H]-silodosin is known asa selective antagonist of α_(1A)- and α_(1L)-subtypes both.

As shown by the competitive curve in (a) of FIG. 2, most (88% or higher)of receptors in the α_(1L) cells exhibit low sensitivity to prazosin(pK_(i)=7.6). Further, the receptors are similarly low in sensitivity toRS-17053, and have high sensitivity to tamsulosin. Further, thereceptors in the α_(1L) cells exhibit only low sensitivity to BMY 7378(Table 2). On the other hand, the sensitivities (pK_(i) values) of theα_(1A) cells, which were used as a control group, to prazosin (FIG. 2),tamsulosin, and RS-17053 are as high as 9.5, 9.9, and 8.8, respectively,which correspond to the α_(1A) properties hitherto reported (Table 2).

TABLE 2 Pharmacologic Properties of α_(1L)-adrenergic Receptors in TwoCell Lines and Human Prostate Human Drug α_(1L) Cells α_(1A) CellsProstate [³H]-silodosin (pK_(D)) 9.1 9.7 9.5 Prazosin 7.6 9.5 8.3Tamsulosin 9.3 9.9 10.0 RS-17053 <6.0 8.8 6.6 BMY 7378 <6.0 6.5 5.9

In this table, the inhibition constants (Kr) of competitive drugs at[³H]-silodosin binding sites in the α_(1L) cells, the α_(1A) cells, andthe human prostate tissue segments are shown as −log K_(i) (pK_(i)).However, the value of [³H]-silodosin exhibits a pK_(D) value(dissociation equilibrium constant) calculated from the saturationbinding curve. The value is an average of three to four examples.

Further, (b) of FIG. 2 shows results obtained by examining sensitivityto prazosin in the homogenates of au. cells and the homogenates ofα_(1A) cells. In the homogenates, the α_(1L) cells exhibited as highsensitivity to prazosin as the α_(1A) cells. This fact coincides wellwith the results that the α_(1L) receptors in prostate and braindisappeared upon tissue homogenization and that the pharmacologicalprofile was converted to α_(1A) phenotype.

Binding affinities for various drugs of α₁-adrenergic receptors inα_(1L) and a 1A cells and human prostate are summarized in Table 2. Theα_(1L) cells, in which the ADRA1A gene and the CRELD1α gene wereco-expressed, differ in receptor properties from a 1A receptors, andcoincides well in pharmacologic properties with α_(1L) receptorsreported in human prostates and the like. Therefore, unlike the α_(1A)cells, the α_(1L) cells are considered as a cell line having expressedα_(1L) receptors predominantly.

4. Intracellular Ca²⁺ Measurement Experiment

Furthermore, in order to completely get rid of the influence of CRELD1α,which might have been endogenously expressed in the α_(1A) cells, theRNAi method was used to produce cells in which CRELD1α expression hadbeen knocked down (such cells being hereinafter referred to as “KDcells”. In order to inhibit gene expression with the RNAi method, avector was produced by inserting an oligonucleotide (sense sequence;GATCCAGGCGACTTAGTGTTCACCTTCAAGAGAGGTGAACACTAAGTCGCCTTTA: SEQ ID NO: 5,antisense sequence;AGCTTAAAGGCGACTTAGTGTTCACCTCTCTTGAAGGTGAACACTAAGTCGCCTG: SEQ ID NO: 6)containing a coding region of mRNA of chinese hamster CRELD1α into acommercially-available pSilencer™4.1-CMV hygro (Ambion, Inc.) accordingto the instruction manual therefor, and then transfected into the α_(1A)cells in the same procedure as described above. The Ca²⁺ response tonoradrenaline in the KD cells was examined according to a fluorescentphotometric method with Fura-2, which is a fluorescent dye. The pEC₅₀value of the Ca²⁺ response to noradrenaline in the KD cells was 7.9. Onthe other hand, when the cells were treated in advance with prazosin10⁻⁸ M for two minutes, the dose-response curve of Ca²⁺ response tonoradrenaline shifted rightward, and the pK_(B) value of prazosin wascalculated to be 9.3. This shows that the Ca²⁺ response to noradrenalinein the KD cells coincides with the properties of the α_(1A)-subtype.

Thus, when the expression of endogenous CRELD1α in a CHO cell having agene of α_(1A)-subtype expressed therein was inhibited by the RNAimethod, α_(1A) receptor properties were perfectly exhibited. It shouldbe noted that there have been some experiments where the receptorfunctions, such as Ca²⁺ response, of α_(1A)-subtype were examined byusing CHO cells in which a gene of the receptor had been expressed.However, there has not necessarily been an agreement in the receptorproperties obtained as a result of those experiments.

Use of the present invention makes it possible to clarify a GPCR whoseentity has been unknown and thereby provide treatment for any diseaseassociated with the receptor, and also makes it possible to modify theligand affinity of a GPCR.

The embodiments and concrete examples of implementation discussed in theforegoing detailed explanation serve solely to illustrate the technicaldetails of the present invention, which should not be narrowlyinterpreted within the limits of such embodiments and concrete examples,but rather may be applied in many variations within the spirit of thepresent invention, provided such variations do not exceed the scope ofthe patent claims set forth below.

INDUSTRIAL APPLICABILITY

It is now clear that α_(1L)-AR is a functional receptor and a maintarget for therapeutic drugs in lower urinary tract system. Thus, thepresent α_(1L)-AR expression cells, developed by the inventors, shouldbe considered to be extremely useful in drug development for urinarydisturbance in patients with benign prostatic hyperplasia and forurinary incontinence. Further, the α_(1L)-AR expression cells areconsidered to be extremely useful as a method for screening therapeuticdrugs for any disease associated with α_(1L)-AR.

What is claimed is:
 1. A protein complex of a GPCR and a polypeptide,the polypeptide being: (A) a polypeptide consisting of an amino-acidsequence of SEQ ID NO: 1; (B) a polypeptide (i) consisting of anamino-acid sequence of SEQ ID NO: 1 with a deletion, insertion,substitution, or addition of one or several amino acids, and (ii) havingactivity to modify ligand affinity of a GPCR with which the polypeptidehas formed a complex; (C) a polypeptide encoded by a polynucleotideconsisting of a nucleotide sequence of SEQ ID NO: 2; (D) a polypeptide(i) encoded by a polynucleotide consisting of a nucleotide sequence ofSEQ ID NO: 2 with a deletion, insertion, substitution, or addition ofone or several nucleotides, and (ii) having activity to modify ligandaffinity of a GPCR with which the polypeptide has formed a complex; (E)a polypeptide (i) encoded by a polynucleotide capable of hybridizingunder stringent conditions with a polynucleotide consisting of asequence complementary to a nucleotide sequence of SEQ ID NO: 2 and (ii)having activity to modify ligand affinity of a GPCR with which thepolypeptide has formed a complex; or (F) a polypeptide (i) coded for bya polynucleotide having a sequence identity of 70% or higher with apolynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and(ii) having activity to modify ligand affinity of a GPCR with which thepolypeptide has formed a complex.
 2. The protein complex as set forth inclaim 1, wherein the GPCR is an adrenergic receptor, a dopaminereceptor, a muscarinic receptor, or an endothelin receptor.
 3. Theprotein complex as set forth in claim 2, wherein the adrenergic receptoris α₁-receptor or a β₁-receptor.
 4. The protein complex as set forth inclaim 3, wherein the α₁-receptor is an α_(1A)-receptor.
 5. The proteincomplex as set forth in claim 2, wherein the dopamine receptor is a D2receptor.
 6. A lipid membrane containing a protein complex as set forthin claim
 1. 7. A method for producing a lipid membrane as set forth inclaim 6, comprising the step of causing a GPCR and a polypeptide tocoexist on a lipid membrane, the polypeptide being: (A) a polypeptideconsisting of an amino-acid sequence of SEQ ID NO: 1; (B) a polypeptide(i) consisting of an amino-acid sequence of SEQ ID NO: 1 with adeletion, insertion, substitution, or addition of one or several aminoacids, and (ii) having activity to modify ligand affinity of a GPCR withwhich the polypeptide has formed a complex; (C) a polypeptide encoded bya polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2;(D) a polypeptide (i) encoded by a polynucleotide consisting of anucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,substitution, or addition of one or several nucleotides, and (ii) havingactivity to modify ligand affinity of a GPCR with which the polypeptidehas formed a complex; (E) a polypeptide (i) encoded by a polynucleotidecapable of hybridizing under stringent conditions with a polynucleotideconsisting of a sequence complementary to a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex; or (F) a polypeptide(i) coded for by a polynucleotide having a sequence identity of 70% orhigher with a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex.
 8. A transformantexpressing a protein complex as set forth in claim
 1. 9. A method forproducing a transformant as set forth in claim 8, comprising the step ofcoexpressing a GPCR and a polypeptide in a cell, the polypeptide being:(A) a polypeptide consisting of an amino-acid sequence of SEQ ID NO: 1;(B) a polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO:1 with a deletion, insertion, substitution, or addition of one orseveral amino acids, and (ii) having activity to modify ligand affinityof a G-protein-coupled receptor with which the polypeptide has formed acomplex; (C) a polypeptide encoded by a polynucleotide consisting of anucleotide sequence of SEQ ID NO: 2; (D) a polypeptide (i) encoded by apolynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 witha deletion, insertion, substitution, or addition of one or severalnucleotides, and (ii) having activity to modify ligand affinity of aGPCR with which the polypeptide has formed a complex; (E) a polypeptide(i) encoded by a polynucleotide capable of hybridizing under stringentconditions with a polynucleotide consisting of a sequence complementaryto a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity tomodify ligand affinity of a GPCR with which the polypeptide has formed acomplex; or (F) a polypeptide (i) coded for by a polynucleotide having asequence identity of 70% or higher with a polynucleotide consisting of anucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modifyligand affinity of a GPCR with which the polypeptide has formed acomplex. 10.-12. (canceled)
 13. A method for producing a transformantexpressing a G-protein-coupled receptor having modified ligand affinity,comprising the step of inhibiting expression of a polypeptide in a cellin which a GPCR has been expressed, the polypeptide being: (A) apolypeptide consisting of an amino-acid sequence of SEQ ID NO: 1; (B) apolypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1with a deletion, insertion, substitution, or addition of one or severalamino acids, and (ii) having activity to modify ligand affinity of aGPCR with which the polypeptide has formed a complex; (C) a polypeptideencoded by a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2; (D) a polypeptide (i) encoded by a polynucleotide consistingof a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,substitution, or addition of one or several nucleotides, and (ii) havingactivity to modify ligand affinity of a GPCR with which the polypeptidehas formed a complex; (E) a polypeptide (i) encoded by a polynucleotidecapable of hybridizing under stringent conditions with a polynucleotideconsisting of a sequence complementary to a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex; or (F) a polypeptide(i) coded for by a polynucleotide having a sequence identity of 70% orhigher with a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex.
 14. The method as setforth in claim 13, the polypeptide is an endogenous protein.
 15. Themethod as set forth in claim 13, wherein the step of inhibiting theexpression of the polypeptide is performed according to an RNAi method.16. The method as set forth in claim 15, wherein the RNAi method isperformed by inserting an oligonucleotide consisting of a nucleotidesequence of SEQ ID NO:
 5. 17. The method as set forth in claim 13,wherein the cell is a transformant expressing an exogenous GPCR.
 18. Amethod for modifying ligand affinity of a GPCR, comprising the step ofinhibiting expression of a polypeptide in a cell in which the GPCR hasbeen expressed, the polypeptide being: (A) a polypeptide consisting ofan amino-acid sequence of SEQ ID NO: 1; (B) a polypeptide (i) consistingof an amino-acid sequence of SEQ ID NO: 1 with a deletion, insertion,substitution, or addition of one or several amino acids, and (ii) havingactivity to modify ligand affinity of a GPCR with which the polypeptidehas formed a complex; (C) a polypeptide encoded by a polynucleotideconsisting of a nucleotide sequence of SEQ ID NO: 2; (D) a polypeptide(i) encoded by a polynucleotide consisting of a nucleotide sequence ofSEQ ID NO: 2 with a deletion, insertion, substitution, or addition ofone or several nucleotides, and (ii) having activity to modify ligandaffinity of a GPCR with which the polypeptide has formed a complex; (E)a polypeptide (i) encoded by a polynucleotide capable of hybridizingunder stringent conditions with a polynucleotide consisting of asequence complementary to a nucleotide sequence of SEQ ID NO: 2 and (ii)having activity to modify ligand affinity of a GPCR with which thepolypeptide has formed a complex; or (F) a polypeptide (i) coded for bya polynucleotide having a sequence identity of 70% or higher with apolynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and(ii) having activity to modify ligand affinity of a GPCR with which thepolypeptide has formed a complex.
 19. A method for screening an agonistfor or antagonist against a GPCR having modified ligand affinity,comprising the steps of: inhibiting expression of a polypeptide in acell in which a GPCR has been expressed; and incubating the celltogether with a candidate factor, the polypeptide being: (A) apolypeptide consisting of an amino-acid sequence of SEQ ID NO: 1; (B) apolypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1with a deletion, insertion, substitution, or addition of one or severalamino acids, and (ii) having activity to modify ligand affinity of aGPCR with which the polypeptide has formed a complex; (C) a polypeptideencoded by a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2; (D) a polypeptide (i) encoded by a polynucleotide consistingof a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,substitution, or addition of one or several nucleotides, and (ii) havingactivity to modify ligand affinity of a GPCR with which the polypeptidehas formed a complex; (E) a polypeptide (i) encoded by a polynucleotidecapable of hybridizing under stringent conditions with a polynucleotideconsisting of a sequence complementary to a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex; or (F) a polypeptide(i) coded for by a polynucleotide having a sequence identity of 70% orhigher with a polynucleotide consisting of a nucleotide sequence of SEQID NO: 2 and (ii) having activity to modify ligand affinity of a GPCRwith which the polypeptide has formed a complex.
 20. The method as setforth in claim 19, further comprising the step of measuring anintracellular Ca²⁺ concentration or the step of measuring metabolism ofintracellular inositol phosphate.